Concerted regulation of skeletal muscle metabolism and contractile properties by the orphan nuclear receptor Nr2f6.

BACKGROUND: The maintenance of skeletal muscle plasticity upon changes in the environment, nutrient supply, and exercise depends on regulatory mechanisms that couple structural and metabolic adaptations. The mechanisms that interconnect both processes at the transcriptional level remain underexplored. Nr2f6, a nuclear receptor, regulates metabolism and cell differentiation in peripheral tissues. However, its role in the skeletal muscle is still elusive. Here, we aimed to investigate the effects of Nr2f6 modulation on muscle biology in vivo and in vitro. METHODS: Global RNA-seq was performed in Nr2f6 knockdown C2C12 myocytes (N = 4-5). Molecular and metabolic assays and proliferation experiments were performed using stable Nr2f6 knockdown and Nr2f6 overexpression C2C12 cell lines (N = 3-6). Nr2f6 content was evaluated in lipid overload models in vitro and in vivo (N = 3-6). In vivo experiments included Nr2f6 overexpression in mouse tibialis anterior muscle, followed by gene array transcriptomics and molecular assays (N = 4), ex vivo contractility experiments (N = 5), and histological analysis (N = 7). The conservation of Nr2f6 depletion effects was confirmed in primary skeletal muscle cells of humans and mice. RESULTS: Nr2f6 knockdown upregulated genes associated with muscle differentiation, metabolism, and contraction, while cell cycle-related genes were downregulated. In human skeletal muscle cells, Nr2f6 knockdown significantly increased the expression of myosin heavy chain genes (two-fold to three-fold) and siRNA-mediated depletion of Nr2f6 increased maximal C2C12 myocyte's lipid oxidative capacity by 75% and protected against lipid-induced cell death. Nr2f6 content decreased by 40% in lipid-overloaded myotubes and by 50% in the skeletal muscle of mice fed a high-fat diet. Nr2f6 overexpression in mice resulted in an atrophic and hypoplastic state, characterized by a significant reduction in muscle mass (15%) and myofibre content (18%), followed by an impairment (50%) in force production. These functional phenotypes were accompanied by the establishment of an inflammation-like molecular signature and a decrease in the expression of genes involved in muscle contractility and oxidative metabolism, which was associated with the repression of the uncoupling protein 3 (20%) and PGC-1α (30%) promoters activity following Nr2f6 overexpression in vitro. Additionally, Nr2f6 regulated core components of the cell division machinery, effectively decoupling muscle cell proliferation from differentiation. CONCLUSIONS: Our findings reveal a novel role for Nr2f6 as a molecular transducer that plays a crucial role in maintaining the balance between skeletal muscle contractile function and oxidative capacity. These results have significant implications for the development of potential therapeutic strategies for metabolic diseases and myopathies.

Antioxidant effects of LEDT in dystrophic muscle cells: involvement of PGC-1α and UCP-3 pathways.

PURPOSE: Reactive oxygen species and mitochondrial dysfunction play a crucial role in the pathophysiology of Duchenne muscular dystrophy (DMD). The light-emitting diode therapy (LEDT) showed beneficial effects on the dystrophic muscles. However, the mechanisms of this therapy influence the molecular pathways in the dystrophic muscles, particularly related to antioxidant effects, which still needs to be elucidated. The current study provides muscle cell-specific insights into the effect of LEDT, 48 h post-irradiation, on oxidative stress and mitochondrial parameters in the dystrophic primary muscle cells in culture. METHODS: Dystrophic primary muscle cells were submitted to LEDT, at multiple wavelengths (420 nm, 470 nm, 660 nm and 850 nm), 0.5 J dose, and evaluated after 48 h based on oxidative stress markers, antioxidant enzymatic system and biogenesis, and functional mitochondrial parameters. RESULTS: The mdx muscle cells treated with LEDT showed a significant reduction of H2O2 production and 4-HNE, catalase, SOD-2, and GR levels. Upregulation of UCP3 was observed with all wavelengths while upregulation of PGC-1α and a slight upregulation of electron transport chain complexes III and V was only observed following 850 nm LEDT. In addition, the mitochondrial membrane potential and mitochondrial mass mostly tended to be increased following LEDT, while parameters like O2·- production tended to be decreased. CONCLUSION: The data shown here highlight the potential of LEDT as a therapeutic agent for DMD through its antioxidant action by modulating PGC-1α and UCP3 levels.

LED therapy plus idebenone treatment targeting calcium and mitochondrial signaling pathways in dystrophic muscle cells.

Intracellular calcium dysregulation, oxidative stress, and mitochondrial dysfunction are some of the main pathway contributors towards disease progression in Duchenne muscular dystrophy (DMD). This study is aimed at investigating the effects of light emitting diode therapy (LEDT) and idebenone antioxidant treatment, applied alone or together in dystrophic primary muscle cells from mdx mice, the experimental model of DMD. Mdx primary muscle cells were submitted to LEDT and idebenone treatment and evaluated for cytotoxic effects and calcium and mitochondrial signaling pathways. LEDT and idebenone treatment showed no cytotoxic effects on the dystrophic muscle cells. Regarding the calcium pathways, after LEDT and idebenone treatment, a significant reduction in intracellular calcium content, calpain-1, calsequestrin, and sarcolipin levels, was observed. In addition, a significant reduction in oxidative stress level markers, such as H2O2, and 4-HNE levels, was observed. Regarding mitochondrial signaling pathways, a significant increase in oxidative capacity (by OCR and OXPHOS levels) was observed. In addition, the PGC-1α, SIRT-1, and PPARδ levels were significantly higher in the LEDT plus idebenone treated-dystrophic muscle cells. Together, the findings suggest that LEDT and idebenone treatment, alone or in conjunction, can modulate the calcium and mitochondrial signaling pathways, such as SLN, SERCA 1, and PGC-1α, contributing towards the improvement of the dystrophic phenotype in mdx muscle cells. In addition, data from the LEDT plus idebenone treatment showed slightly better results than those of each separate treatment in terms of SLN, OXPHOS, and SIRT-1.